Overview

  • Product nameAnti-beta Actin antibodySee all beta Actin primary antibodies ...
  • Description
    Rabbit polyclonal to beta Actin
  • Tested applicationsIHC-Fr, IP, WB, ICC, Flow Cyt, IHC-FrFl, IHC-P, ICC/IF, ELISA more details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Cow, Dog, Human, Pig, Xenopus laevis, Fish, Monkey, Rhesus monkey, Chinese Hamster

    Does not react with

    Zebrafish
  • Immunogen

    Synthetic peptide derived from within residues 1 - 100 of Human beta Actin.

    (Peptide available as ab13772.)

  • Positive control
    • This antibody gave a positive signal in Mouse Brain tissue lysate as well as the following whole cell lysates: HeLa; A431; HEK293; NIH3T3; PC12; Yeast Extract.

Properties

Applications

Our Abpromise guarantee covers the use of ab8227 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IHC-Fr Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/1000 - 1/5000. Can be blocked with Human beta Actin peptide (ab13772).
ICC Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.
IHC-FrFl Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 µg/ml.
ELISA 1/1000.

Target

Anti-beta Actin antibody images

  • All lanes : Anti-beta Actin antibody (ab8227) at 0.1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 3 : Liver (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Performed under reducing conditions.

    Observed band size : 42 kDa (why is the actual band size different from the predicted?)


    Exposure time : 1 minute

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution

    Observed band size : 42 kDa (why is the actual band size different from the predicted?)

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Milk before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using ab175781 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/5000 dilution

    Lane 1 : HeLa whole cell extract
    Lane 2 : Yeast cell extract
    Lane 3 : Mouse brain tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/2000 dilution

    Observed band size : 47 kDa (why is the actual band size different from the predicted?)

    Rabbit polyclonal to beta actin (ab8227) at 1/5000. Lane 1: HeLa whole cell extract (20ug); lane 2: yeast cell extract (20ug); lane 3: mouse brain tissue lysate (20ug).

    Developed using Goat anti-rabbit IgG (HRP) (ab6721) at 1/2000.

  • ICC/IF image of ab8227 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab8227, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue).

  • IHC image of beta Actin staining in human liver FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8227, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • ab8227 staining beta Actin in human stomach tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with acetone and then blocked with 5% serum for 1 hour at 23°C followed by incubation with the primary antibody, at 1µg/ml, for 1 hour at 23°C. An undiluted HRP-conjugated goat polyclonal was used as secondary antibody.

    See Abreview

  • ab8227 used in Direct ELISA in NIH 3T3 murine fibroblasts. Primary antibody used at a 1/1000 dilution for 16 hours at 4°C. The secondary antibody used is an AP-conjugated Goat anti-rabbit used at a 1/2000 dilution. A blocking step was performed using 5% BSA for 1 hour at 23°C.

    See Abreview

  • ICC/IF image of ab8227 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8227, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa cells (Human) at 20 µg
    Lane 2 : 3T3 cells (Mouse) at 20 µg
    Lane 3 : Fish Liver at 20 µg
    Lane 4 : Rabbit Liver at 20 µg
    Lane 5 : MDCK cells (Dog) at 20 µg/ml
    Lane 6 : EBTr cells (Cow) at 20 µg
    Lane 7 : SL-29 cells (Chicken) at 20 µg/ml
    Lane 8 : CHO cells (Chinese Hamster) at 20 µg
    Lane 9 : Xenopus embryo at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Observed band size : 41.7 kDa (why is the actual band size different from the predicted?)


    Exposure time : 30 seconds

    Western blot using ab8227 at 1/1000.

    Lysates:
    Lane 1: HeLa cells (Human)
    Lane 2: 3T3 cells (Mouse)
    Lane 3: Fish Liver
    Lane 4: Rabbit Liver
    Lane 5: MDCK cells (Dog)
    Lane 6: EBTr cells (Cow)
    Lane 7: SL-29 cells (Chicken)
    Lane 8: CHO cells (Chinese Hamster)
    Lane 9: Xenopus embryo

    Secondary ab: Rabbit polyclonal to Goat IgG H&L (HRP) ab6741 (1/5000)
    Exposure time: 30 sec
    Lysates at 20µg/lane.
    Expected molecular weight: 41.7kDa.

  • ab8227 staining beta Actin in human LGE neural progenitors by Flow Cytometry. Cells were paraformaldehyde and permeabilized. The sample was incubated with the primary antibody (1/300) for 1 hour at 20°C. A PerCP-conjugated goat anti-rabbit IgG (1/800) was used as the secondary antibody.
    Gating Strategy: Isotype population (shown in white).

    See Abreview

References for Anti-beta Actin antibody (ab8227)

This product has been referenced in:
  • McGrath-Morrow SA  et al. Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status. Cytokine 65:4-9 (2014). Mouse . Read more (PubMed: 24139870) »
  • Alvarez P  et al. Ectopic endometrium-derived leptin produces estrogen-dependent chronic pain in a rat model of endometriosis. Neuroscience 258:111-20 (2014). Rat . Read more (PubMed: 24239717) »

See all 219 Publications for this product

Product Wall

Thank you for contacting us.

I have had the opportunity to discuss our QC methods regarding this product witht eh lab. Ab17721 is batch tested by us in WB in HeLa, Jurkat and NIH3T3 lysates and also in Immunofluorescence in HeLa cells. Each n...

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Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - Perm wash buffer
Sample Human Cell (LGE Neural Progenitors)
Specification LGE Neural Progenitors
Gating Strategy isotype population (shown in white)
Preparation Cell harvesting/tissue preparation method: 5min collagenase followed by 5mins of accutase
Sample buffer: 5% FBS wash
Username

Dr. Adam Johnson

Verified customer

Submitted Apr 18 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12%)
Sample Rat Tissue lysate - whole (ovaries)
Specification ovaries
Blocking step Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Username

Mrs. Henda Nabli

Verified customer

Submitted Oct 01 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (human fibroblast cells)
Specification human fibroblast cells
Blocking step Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5µg/mL · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Aug 14 2013

Application Western blot
Loading amount 1e+006 cells
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (B cells and Macrophages)
Specification B cells and Macrophages
Treatment Selenium or Rapamycin
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 12 2013

Abreviews
Application Western blot
Loading amount 15 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (Ad293)
Specification Ad293
Blocking step BSA as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted May 29 2013

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Tissue lysate - whole (epidermis)
Specification epidermis
Blocking step BSA as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted May 29 2013

Application Western blot
Sample Human Cell lysate - whole cell (Daudi (Human Burkitt's lymphoma cell line))
Loading amount 20 µg
Specification Daudi (Human Burkitt's lymphoma cell line)
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Dr. Elena Kashuba

Verified customer

Submitted Apr 05 2013

Application Western blot
Sample Human Cell lysate - whole cell (Human mast cell line)
Loading amount 20 µg
Specification Human mast cell line
Treatment tricho
Gel Running Conditions Reduced Denaturing (gel 10%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Ms. na yeong gu

Verified customer

Submitted Apr 02 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Rat Cell (Rat Astrocytes culture)
Specification Rat Astrocytes culture
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% TritonX in 0.1% PBS
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C
Username

Dr. Ruma Raha-Chowdhury

Verified customer

Submitted Dec 19 2012

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